Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture

Plyusnina, Angelina; Plyusnin, Alexander

The genetic swarm of S RNA molecules from the recTULV is represented almost exclusively by the variant with a single break point located between nt332 and nt368. The proportion of the dominant variant is larger in the passaged recTULV (13 of 14 cDNA clones analyzed, or 93%) than in the freshly formed mixture of recS RNAs (12 of 20 cDNA clones, or 60%) [11] . Thus, recTULV already represents a product of a micro-evolutionary play, in which the best-fit variant has been selected from the initial mixture of recS RNA. Whether this resulted from higher frequency of recombination through the "hot-spot" located between nt332 and nt368 or from the swift elimination of all other products of random recombination due to their lower fitness (the situation reported for polio-and coronaviruses [22, 23] ), or both, remains unclear. We favor the first explanation as the modeling of the S RNA folding suggests formation of a relatively long hairpin-like structure within the recombination "hot-spot" (Fig. 3) . Secondary structure elements of this kind, which might present obstacles for sliding of the viral RNA polymerase along the template, were suggested as promoters for the template- switching in the early studies on polioviruses [22] and considered a crucial prerequisite for recombination [25, 24] . The hairpin in TULV plus-sense S RNA (Fig. 3) is formed by the almost perfect inverted repeat that includes nt 344 to 374. In the minus-sense RNA, the structure is slightly weaker due to the fact that two non-canonical G:U base pairs presented in the plus-sense RNA occur as nonpairing C/A bases in the minus-sense RNA. Interestingly, in Puumala hantavirus, a hairpin-like structure formed by a highly conserved inverted repeat in the 3'-noncoding region of the S segment seems to be involved in recombi-nation events, leading, however, to the deletion of the hairpin-forming sequences (A. Plyusnin, unpublished observations). The role of RNA folding in hantavirus recombination awaits further investigation.